Summary A significant number of Americans work non-traditional schedules and suffer the adverse health effects of a disrupted circadian timing system. The master mammalian circadian clock, located in the suprachiasmatic nucleus (SCN) maintains the proper phase relationship between circadian clocks located in tissues throughout the body and entrains the circadian system to the environment. The SCN is composed of individual neuronal oscillators coupled by intercellular communication into a neural network that generates a robust and precise rhythm. The long-term goal of our research is to understand the intercellular signaling mechanisms that couple SCN neurons into a neural network that generates circadian rhythms. GABAergic neurotransmission is a fundamental component of the SCN neural network and changing the strength and polarity of postsynaptic GABA responses modifies the activity of the SCN, and ultimately circadian rhythmicity. GABA serves as a desynchronizing signal under equilibrium conditions and a synchronizing signal when the SCN neural network has been modified by environmental light. GABA acts on synaptic GABA(A) receptors to mediate fast signaling between SCN neurons and on extrasynaptic GABA(A) receptors to activate a tonic GABA(A) current that modulates the activity of individual SCN neurons and communicates the level of network activity to adjacent synapses. We hypothesize that two membrane transporter families play critical roles in the regulation of the circadian activity of GABA neurotransmission in the SCN. The GABA transporters GAT-1 and GAT-3 regulate the amount and duration of neurotransmitter GABA in the extrasynaptic space and the magnitude of the tonic current. In the SCN, the GABA transporters are only expressed in astrocytes suggesting that astrocytes play a vital, but as of yet undetermined role in regulating the physiological actions of GABA in the SCN network. In the adult SCN, GABA serves as both an inhibitory and excitatory neurotransmitter although the physiological significance of this change in the polarity of GABA neurotransmission remains unknown. The chloride cotransporters of the sodium-potassium-chloride (NKCC) and potassium-chloride (KCC) families control the intracellular Cl- concentration and the polarity and magnitude of the GABA(A) receptor-mediated currents. We propose that the circadian clock uses the intracellular second messenger systems WNK-SPAK kinases, Ca2+- activated kinases, and cyclic AMP-activated kinases to regulate the activity of the Cl- transporters. The goal of this application is to understand better the mechanisms regulating GABAergic signaling and how GABA- mediated signaling contributes to the generation of circadian timing signals in the SCN. To accomplish this goal, we will use single cell electrophysiological and imaging techniques together with transgenic mouse models to study GABAergic neurotransmission in identified SCN neurons. Enhanced knowledge of the intercellular signaling mechanisms mediated by GABA will increase our ability to manipulate the circadian clock and reduce the symptoms experienced by patients with circadian-based disorders.